Our goal is to understand how a cell can coordinate the expression of its genes during balanced growth as well as during transient nutritional impoverishment. Our focus continues on the role played by the regulatory nucleotide, guanosine 3',5'-bispyrophosphate (ppGpp) in mediating these cellular responses in E. coli. A few years ago, we began characterizing the genes and the regulatory elements governing the metabolism of ppGpp, This information has been exploited to disentangle cause and effect relationships by artificially manipulating intracellular ppGpp levels. Previous reports have described the cloning and sequencing of the relA gene (which catalyses ribosome-dependent ppGpp synthesis during aminoacyl tRNA deprivation), the spoT gene (which catalyses ppGpp degradation to GDP), and a low level residual synthetic activity that persists despite deletion of the entire relA gene. This year, we have identified genes in the spoT operon that give a relA gene-dependent phenotype when interrupted as well as one that is a probable subunit of RNA polymerase, the omega subunit. We have also discovered cis-dominant complementation functions associated with mutations of these genes that suggests complex interactions at a regulatory or subunit association level. Most surprisingly, the source of residual synthetic activity has been localized to the spoT operon. We have shown that one of the requirements for this synthetic activity is the spoT gene itself, which normally catalyzes ppGpp degradation. We have found that artificial induction of high levels of ppGpp under nutritionally sufficient conditions stops cellular growth and exerts regulatory effects on gene expression in a manner that is very similar to the responses seen when ppGpp is naturally induced during nutritional stress. Thus, stopping growth during nutritional starvation might not be due to the starvation per se but instead to a ppGpp-sensitive step that has protective value for the cell. We have begun the isolation of ppGpp-resistant mutants defective in this step.